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PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial Pamps

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PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial Pamps PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs Frances R. Wadelin, Joel Fulton, Hilary M. Collins, Nikolaos Tertipis, Andrew Bottley, Keith A. Spriggs, Franco H. Falcone, David M. Heery* School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, United Kingdom Abstract Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNc). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNc resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNc also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNc treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi. Citation: Wadelin FR, Fulton J, Collins HM, Tertipis N, Bottley A, et al. (2013) PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs. PLoS ONE 8(2): e58052. doi:10.1371/journal.pone.0058052 Editor: William R. Abrams, New York University, United States of America Received January 5, 2012; Accepted January 30, 2013; Published February 27, 2013 Copyright: ß 2013 Wadelin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Leukaemia Lymphoma Research via a clinical training fellowship to FRW (07064). JF was funded by the Royal Pharmaceutical Society of Great Britain via an Academic Excellence Award to DMH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction pathogenic bacteria [16], although until recently there has been little insight into its functions in mammalian cells. Nuclear PRAME/MAPE/OIP4 is an atypical cancer-testis antigen localised PRAME has been implicated in transcriptional repres- whose expression is associated with leukaemias and a large sion via association with retinoic acid receptor complexes [17]. proportion of solid tumours (reviewed in [1]). Unlike other More recently, while this study was in progress, PRAME was cancer-testis antigens whose expression is restricted to testis, the shown to be a chromatin-associated protein enriched at nuclear PRAME gene shows low level expression in other normal tissues factor Y (NFY) target genes, in association with Elongin and including endometrium, ovary and placenta [2]. The restricted Cullin-2 proteins [18]. However, a large proportion of endog- expression pattern of PRAME in normal tissues and its enous PRAME protein is observed in the cytoplasmic compart- overexpression in tumours renders it a useful marker of minimal ment in different cell lines [1,19]. Given its reported interaction residual disease after chemotherapy, and an attractive target for with bacterial outer membrane proteins [16] and the rapid immunotherapy, particularly in acute myeloid leukaemia (AML) evolution of the PRAME multigene family in humans [15], and chronic myeloid leukaemia (CML) [2,3,4,5,6,7,8,9,10,11,12]. similar to the Nacht, LRR, PYD domain (NALP) gene family In PRAME-negative leukaemias the gene can be induced by [20], we hypothesised that PRAME might be regulated by demethylating agents [2,13,14], but little else is known regarding signalling pathways activated in proinflammatory responses. We pathways that regulate PRAME expression. therefore set out to investigate whether PRAME expression is PRAME is a member of a rapidly evolving multigene family, modulated by signalling molecules such as IFNc or microbial and encodes a leucine-rich repeat (LRR) protein sharing PAMPs. We also endeavoured to isolate PRAME-interacting structural similarity with Toll-like receptors [1,15]. The extensive proteins, to provide further insight into its cellular functions. duplication rate of PRAME and other cancer testis antigen genes is suggestive of roles in chemosensing, reproduction or immunity [15]. PRAME was originally identified in a yeast two-hybrid screen for proteins that bind outer membrane proteins of PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e58052 Function and Regulation of PRAME Results the cytoplasm of these cells within 24 hrs of transfection (Fig. 2A). Staining with a specific antibody recognising PRAME confirmed Upregulation of PRAME expression by IFNc and bacterial the identity of the recombinant PRAME-EGFP and PRAME- PAMPs FLAG constructs, in contrast to neighbouring non-transfected cells PRAME protein contains a series of leucine-rich repeats similar which showed negligible endogenous levels of PRAME (Fig. 2A). to those found in the LRR protein family and is predicted to be In contrast to PRAME-EGFP, EGFP alone showed approximately structurally similar to human Toll-like receptors (TLRs) [1,15]. equal distribution in the nucleus and cytoplasm whereas co- Unlike the membrane-associated TLRs, PRAME is an intracel- transfected PRAME-FLAG was predominantly cytoplasmic lular protein found in both the nuclear and cytoplasmic (Fig. 2A, lower panels). These observations are consistent with compartments [1]. TLRs function in innate immunity as sensors suggestions that PRAME is likely to have both nuclear and of microbial PAMPs or other ligands, and their expression is cytoplasmic functions, although the distribution between these regulated by these molecules and also by IFNc, a proinflammatory compartments may vary in different cell types and conditions. cytokine produced in response to infection. We therefore assessed To determine whether exposure to bacterial PAMPs/IFNc had whether PRAME expression in leukaemic cells such as HL60 might any effect on the subcellular localisation of PRAME, we be modulated by exposure to lipopolysaccharide (LPS) and IFNc, performed immunofluorescence staining of cells exposed to these either as single inducing agents or in combination. HL60 agents, or controls. A longer treatment time (4 hours) was used to leukaemic cells have low levels of PRAME transcripts [1], and allow for expression of PRAME proteins in the cells. As shown in are known to express TLR2 and TLR4 [21]. Treatment of HL60 Fig. 2B, treatment of HL60 cells with LPS/IFNc (middle panels) cells with IFNc or LPS alone did not significantly alter the or PGN/IFNc (lower panels) for 4 hours resulted in localisation of expression of PRAME (as determined by RT-qPCR – data not PRAME to large cytoplasmic foci. A similar effect was observed in shown). However, as shown in Fig. 1A, a strong increase in the leukaemic cell line U937 in response to LPS/IFNc treatment PRAME transcript levels was observed within 1 hour of combined (Fig. 2C). Quantitation of PRAME-containing foci in LPS/IFNc- treatment with LPS and IFNc. This increase was transient as treated and or control cells revealed a major change in the PRAME levels were lower at 4 hours post-treatment, and restored subcellular distribution of PRAME in response to LPS/IFNc to background levels within 24 hours, suggesting that turnover of treatment (Fig. 2E&F). PRAME mRNA may be relatively rapid. The induction of PRAME The polarised distribution of the PRAME-containing foci, by LPS/IFNc was inhibited by pre-treating cells with actinomycin suggested that these structures may represent the trans Golgi D for one hour prior to addition of LPS/IFNc (Fig. 1B), indicating network, and staining of HL60 cells with the 58K Golgi marker that the increase in PRAME levels is due to de novo transcription confirmed that these structures appear to be linked to the Golgi rather than altered stability of the transcripts. network and show considerable colocalisation with PRAME To examine whether LPS/IFNc results in increased translation (Fig. 2D). We noted that the nuclei of LPS/IFNc treated HL60 of PRAME transcripts, polysome gradients were prepared from and U937 cells appeared to undergo a morphological change extracts of HL60 cells treated or untreated with LPS and IFNc.As resulting in ‘kidney’ shaped nuclei being visible after 4 hours shown in Fig. 1C, within 4 hours post-treatment with LPS/IFNc, treatment (Fig. 2B&C), and that this phenotype was prevalent in PRAME transcripts were substantially enriched in polysome many of the treated cells, but less obvious in controls (Fig. 2E). fractions, as confirmed by densitometry (lower panels). In contrast, a control transcript (b-actin) showed little change. These results PRAME and the 58K Golgi marker accumulated at the apex of indicate that upregulation of PRAME gene expression by LPS/ the invaginated region (Fig. 2D, lower panels). Thus, we conclude IFNc in HL60 cells may be achieved not only through increased that in HL60 and U937 cells, cytoplasmic PRAME proteins are transcription, but also effects on translation of PRAME transcripts. targeted to the Golgi network following exposure to LPS/IFNc.
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